structure of a mutant form of proliferating cell nuclear antigen that blocks translesion dna...

Structure of a Mutant Form of Proliferating Cell Nuclear Antigen That BlocksTranslesion DNA Synthesis†,‡

Bret D. Freudenthal,§ S. Ramaswamy,§ Manju M. Hingorani,| and M. Todd Washington*,§

Department of Biochemistry, UniVersity of Iowa College of Medicine, Iowa City, Iowa 52242-1109, and Molecular Biology andBiochemistry Department, Wesleyan UniVersity, Middletown, Connecticut 06459

ReceiVed September 17, 2008; ReVised Manuscript ReceiVed October 31, 2008

ABSTRACT: Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein that functions as a slidingclamp during DNA replication. Several mutant forms of PCNA that block translesion DNA synthesishave been identified in genetic studies in yeast. One such mutant protein (encoded by the reV6-1 allele)is a glycine to serine substitution at residue 178, located at the subunit interface of PCNA. To improveour understanding of how this substitution interferes with translesion synthesis, we have determined theX-ray crystal structure of the PCNA G178S mutant protein. This substitution has little effect on the structureof the domain in which the substitution occurs. Instead, significant, local structural changes are observedin the adjacent subunit. The most notable difference between mutant and wild-type structures is in asingle, extended loop (comprising amino acid residues 105-110), which we call loop J. In the mutantprotein structure, loop J adopts a very different conformation in which the atoms of the protein backbonehave moved by as much as 6.5 Å from their positions in the wild-type structure. To improve ourunderstanding of the functional consequences of this structural change, we have examined the ability ofthis mutant protein to stimulate nucleotide incorporation by DNA polymerase eta (pol η). Steady statekinetic studies show that while wild-type PCNA stimulates incorporation by pol η opposite an abasicsite, the mutant PCNA protein actually inhibits incorporation opposite this DNA lesion. These resultsshow that the position of loop J in PCNA plays an essential role in facilitating translesion synthesis.

DNA damage in the template strand blocks replication byclassical DNA polymerases, which are involved in normalDNA replication and repair. To overcome these replicationblocks, cells employ several nonclassical DNA polymerasesthat are capable of replicating through template DNA lesionsin a process called translesion DNA synthesis (1-3). Onesuch enzyme is eukaryotic DNA polymerase eta (pol1 η),which is a 71 kDa monomeric protein encoded by the RAD30gene in yeast (4). pol η functions in the replication of a fewtypes of DNA lesions, including thymine dimers (4-6) and8-oxoguanines (7, 8). Deletion of the RAD30 gene in yeastleads to an increase in the level of ultraviolet (UV) radiation-induced mutagenesis (9-11), and in humans, inactivationof pol η is responsible for the variant form of xerodermapigmentosum (XPV) (12, 13), which results in greater cancer

susceptibility. Another nonclassical DNA polymerase ineukaryotes is DNA polymerase zeta (pol �), which iscomprised of a 173 kDa catalytic subunit and a 29 kDaaccessory subunit encoded in yeast by the REV3 and REV7genes, respectively (14, 15). pol � functions in the error-prone replication of a wide range of DNA lesions, anddisruptions of the REV3 and REV7 genes result in a drasticdecrease in the frequency of DNA damage-induced mutationsin yeast (16, 17). Moreover, expression of antisense RNAto pol � leads to a reduction in the frequency of UV radiation-induced mutations in human cells (18).

A key factor in translesion synthesis is proliferation cellnuclear antigen (PCNA). PCNA, encoded in yeast by thePOL30 gene, is a ring-shaped, homotrimeric protein that actsas a sliding clamp for classical DNA polymerases (19, 20).Many protein factors involved in DNA replication and repairinteract with PCNA via their PCNA-interacting peptide (PIP)motifs that bind along the interdomain connector loop ofPCNA (21). pol η binds to PCNA in this manner, and thisinteraction is necessary for pol η function in ViVo (22, 23).Moreover, this interaction stimulates the enzymatic activityof pol η in Vitro (22). pol �, although lacking a PIP motif,also interacts with PCNA, and its enzymatic activity isstimulated by PCNA (24).

Several PCNA mutant proteins in yeast have been identi-fied that interfere with translesion synthesis in vivo (25-27).One of these is encoded by the pol30-178 allele (formerlycalled the reV6-1 allele); it encodes a mutant form of PCNAin which Gly-178 is substituted with a serine (25). This amino

† This work was supported by Grant R01GM081433 from theNational Institute of General Medical Sciences.

‡ Atomic coordinates were deposited in the RCSB Protein Data Bankas entry 3F1W.

* To whom correspondence should be addressed: Department ofBiochemistry, 4-403 Bowen Science Building, University of Iowa, IowaCity, IA 52242-1109. Phone: (319) 335-7518. Fax: (319) 335-9570.E-mail: [email protected].

§ University of Iowa College of Medicine.|Wesleyan University.1 Abbreviations: dNTP, deoxynucleoside triphosphate; DTT, dithio-

threitol; EDTA, ethylenediaminetetraacetic acid; MMS, methyl meth-anesulfonate; PCNA, proliferating cell nuclear antigen; PIP, PCNA-interacting peptide; PMSF, phenylmethanesulfonyl fluoride; pol,polymerase; RFC, replication factor C; rmsd, root-mean-square devia-tion; UV, ultraviolet; XPV, xeroderma pigmentosum variant form.

Biochemistry 2008, 47, 13354–1336113354

10.1021/bi8017762 CCC: $40.75 2008 American Chemical SocietyPublished on Web 11/20/2008

acid substitution is at the subunit interface of PCNA, andgenetic studies have shown that translesion synthesis by bothpol η and pol � is completely blocked in cells expressingonly this mutant form of PCNA (25). All other aspects ofDNA replication and repair appear to occur normally in cellsexpressing this PCNA mutant protein (25). Another PCNAmutant protein that blocks translesion synthesis but supportsnormal cell growth is encoded by the pol30-113 allele (26).In this mutant protein, Glu-113 is substituted with a glycine.Interestingly, Glu-113 is also located at the subunit interfaceof PCNA directly opposite from Gly-178 on the neighboringsubunit. On the basis of the fact that these mutant proteinsblock translesion synthesis without interfering with normalDNA replication, the structural changes resulting from theseamino acid substitutions are likely subtle.

To understand the structural and mechanistic basis of thedefect in the PCNA G178S mutant protein’s ability to supporttranslesion synthesis, we determined the X-ray crystalstructure of this mutant protein to a resolution of 2.9 Å. Wefind that the substituted serine side chain forms a newhydrogen bond with the backbone carbonyl of Glu-113 onthe adjacent subunit. This contact results in an extended loopon the adjacent subunit (comprising amino acid residues105-110, which we call loop J), changing its conformationand moving it into an aberrant position that deviates as muchas 6.5 Å from its position in the wild-type structure. Wehave examined the biochemical relevance of this structureby carrying out steady state kinetic studies with a modelnonclassical polymerase, pol η, in the presence of the wild-type and mutant PCNA proteins. We find that while wild-type PCNA stimulates incorporation by pol η opposite anabasic site, the mutant PCNA protein inhibits incorporationopposite this DNA lesion. These findings suggest that theproper conformation of loop J is essential for translesionDNA synthesis.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification. Wild-type and mutantPCNA proteins from Saccharomyces cereVisiae were over-expressed in Escherichia coli Rosetta-2 (DE3) cells harboringpET-11a vectors into which wild-type or mutant PCNA geneswere cloned. PCNA proteins were N-terminally FLAGtagged for rapid purification. Transformed cells were grownat 37 °C in Overnight Express Instant TB Medium (Novagen)for 12 h. Cells were lysed in 50 mM Tris-HCl (pH 7.5), 150mM NaCl, 1 mM PMSF, and 1 mg/mL lysozyme, with aComplete Mini Protease Inhibitor Cocktail tablet (Roche).Following ultracentrifugation, the crude extract was loadedonto a 15 mL resin bed of Anti-FLAG M2 Affinity Gel(Sigma) and purified by following the instructions. The elutedPCNA protein was then run on a Superdex G-75 columnequilibrated with 20 mM Tris-HCl (pH 7.5), 1 mM DTT,and 250 mM NaCl. Yeast pol η and RFC were overexpressedand purified as previously described (28, 29). All purifiedproteins were stored in aliquots at -80 °C.

Crystallization of the PCNA G178S Mutant Protein.Crystallization of the PCNA G178S mutant protein wasperformed manually using the hanging drop method with 4µL drops. To identify ideal crystallization conditions, aninitial screen utilized conditions similar to those whichproduce wild-type PCNA crystals (19). Optimally diffracting

crystals were generated by combining an equal volume ofprotein (30 mg/mL) with a reservoir solution containing 2.06M ammonium sulfate and 0.1 M sodium citrate (pH 5.8).Cubic crystals formed within 16 h at 18 °C.

Data Collection and Structural Determination. PCNAG178S protein crystals were presoaked in a mother liquorcontaining 10% (v/v) glycerol prior to being flash-frozen at100 K. Mounted crystals were subsequently used for datacollection at 100 K at the 4.2.2 synchrotron beamline at theAdvanced Light Source in the Ernest Orlando LawrenceBerkeley National Laboratory. The data were collected witha crystal to detector distance of 150 mm. The data wereanalyzed and scaled using d*trek (30) to a resolution of 2.9Å, and the space group was determined to be P213, whichis the same space group into which the wild-type PCNAprotein crystallizes (19).

We carried out molecular replacement using the knownstructure of wild-type PCNA [Protein Data Bank (PDB) entry1PLQ] and PHASER (31) to produce a model with the P213space group. To remove any structural bias, simulatedannealing was performed using PHENIX (32) prior to anyrefinement. Further structural refinement was executed usingPHENIX and REFMAC5 from the CCP4 package (31).Model building was carried out using Coot and O (34). Thecoordinates have been deposited in the PDB as entry 3F1W.

DNA Substrates. For DNA polymerase activity assays, asynthetic 68-mer oligodeoxynucleotide with the sequence 5′-biotin-GAC GGC ATT GGA TCG ACC TCX AGT TGGTTG GAC GGG TGC GAG GCT GGC TAC CTG CGATGA GGA CTA GC-biotin was used as the template strand(X is an abasic site or a nondamaged guanine). For therunning start abasic bypass assays, a synthetic 31-meroligodeoxynucleotide with the sequence 5′-TCG CAG GTAGCC AGC CTC GCA CCC GTC CAA C was used as aprimer. For the steady state kinetic studies, a synthetic 31-mer oligodeoxynucleotide with the sequence 5′-GGT AGCCAG CCT CGC ACC CGT CCA ACC AAC T was used asa primer. Primer strands were 5′-32P-end-labeled using T4polynucleotide kinase (New England Biolabs) and[γ-32P]ATP (PerkinElmer). Template strands and labeledprimer strands (1 µM each) were annealed in 25 mM Tris-HCl (pH 7.5) and 100 mM NaCl by being heated to 90 °Cfor 2 min and slowly cooled to 22 °C over several hours.Labeled and annealed DNA substrates were stored at 4 °Cfor up to 2 weeks.

Polymerase ActiVity Assays. All experiments were con-ducted in 40 mM Tris-HCl (pH 7.5), 8 mM MgCl2, 150 mMNaCl, 1 mM DTT, and 100 µg/mL bovine serum albumin.Reaction mixtures also contained a 10-fold molar excess ofstreptavidin over DNA to block the ends of the DNA toprevent PCNA dissociation. Wild-type or mutant PCNAproteins were loaded onto the DNA substrates via incubationof 90 nM PCNA (trimer concentration), 20 nM DNA, 25nM RFC, and 500 µM ATP for 5 min at 22 °C. Reactionmixtures also contained various concentrations of dGTP (0to 100 µM for the abasic site template) or dCTP (0 to 5 µMfor the nondamaged template). Reactions were initiated viaaddition of 1 nM pol η and were quenched after 10 min bythe addition of 10 volumes of formamide loading buffer [80%deionized formamide, 10 mM EDTA (pH 8.0), 1 mg/mLxylene cyanol, and 1 mg/mL bromophenol blue]. Extendedprimers (the product) and unextended primers (the substrate)

A PCNA Mutant Protein That Blocks Translesion Synthesis Biochemistry, Vol. 47, No. 50, 2008 13355

were separated on a 15% polyacrylamide sequencing gelcontaining 8 M urea. The intensities of the labeled gel bandswere determined using the InstantImager (Packard). The rateof product formation was graphed as a function of dNTPconcentration, and the Vmax and Km values were obtained fromthe best fit of the data to the MichaelissMenten equationusing SigmaPlot 10.0.

The running start abasic site bypass assay was performedunder conditions identical to those of the steady state kineticstudies except that each dNTP was used at a concentrationof 20 µM. Reactions were quenched after 5, 10, and 15 minby the addition of 10 volumes of formamide loading buffer,and reaction products were visualized on a 15% polyacry-lamide sequencing gel containing 8 M urea.

RESULTS

Several mutant forms of yeast PCNA that are unable tosupport translesion DNA synthesis in vivo have beenidentified in genetic screens (25-27). One of these is thePCNA G178S mutant protein. Yeast strains expressing thismutant form of PCNA have the same phenotype as strainslacking both pol η and pol � (25). To improve ourunderstanding of how this mutant form of PCNA blockstranslesion synthesis, we have determined the X-ray crystalstructure of this mutant protein and have carried outfunctional studies of pol η in the presence of this mutantprotein.

OVerView of the PCNA G178S Protein Structure. We havedetermined the X-ray crystal structure of the PCNA G178Smutant protein to a resolution of 2.9 Å (Table 1). Figure 1Ashows an overview of the structure of the PCNA G178Smutant protein trimer. Each subunit is comprised of twodomains arranged in the trimeric ring in a head-to-tail fashionwith domain A (residues 1-118) of one subunit interactingwith domain B (residues 135-240) of its neighbor. Withineach subunit, domains A and B are linked by the interdomain

connector loop (residues 119-134). No large-scale differ-ences in the structure of the PCNA G178S mutant proteinand the wild-type PCNA protein can be detected.

Figure 1B shows a closer view of the subunit interface ofthe PCNA mutant protein. The G178S substitution is locatedin � strand D2 of domain B of the upper subunit. The sidechain hydroxyl group of the substituted serine forms a newhydrogen bond with the backbone carbonyl of Glu-113 onthe adjacent subunit. The two oxygen atoms are 2.6 Å apart,which is typical for a hydroxyl group-carbonyl grouphydrogen bond. Glu-113 is in � strand I1, which forms anantiparallel � sheet with strand H1. In the mutant proteinstructure, the new hydrogen bond between Ser-178 and Glu-113 alters the trajectory of this antiparallel � sheet. This is

Table 1: Data Collection and Refinement Statistics

Data Collection Statistics

resolution (Å) 29.8-2.9wavelength (Å) 1.072space group P213cell (Å) a ) 123.13, b ) 123.13, c ) 123.13completeness (%) (last shell) 100 (3.0-2.9) (96.8)a

redundancy 10.79 (11.01)a

I/σI 14.8 (2.50)a

Rmerge (%)b 7.3 (60.5)a

Refinement Statistics

resolution range (Å) 19.9-2.9R (%)c 22.6Rfree (%)d 25.4rmsd for bonds (Å) 0.009rmsd for angles (deg) 1.21no. of water molecules 0no. of protein atoms 1981 (254 amino acid residues)Ramachandran analysis (%)

most favored 89.4allowed 10.6

PDB entry 3F1Wa Values in parentheses relate to the highest-resolution shell. b Rmerge

) ∑I - ⟨I⟩/∑I, where I is the observed intensity and ⟨I⟩ is the averageintensity obtained from multiple observations of symmetry-relatedreflections after rejections. c R ) ∑|Fo - Fc|/∑Fo, where Fo and Fc arethe observed and calculated structure factors, respectively. d Rfree isdefined by Brunger (38).

FIGURE 1: Structure of the PCNA G178S mutant protein. (A) Thetrimeric form of the protein is shown with monomeric subunitscolored red, yellow, and blue. The interdomain connecting loop(IDCL), domains A and B, and the G178S substitution are indicatedon one of the subunits. (B) Close-up, side view of the subunitinterface with the Ser-178 substitution of the blue monomer andTyr-114 and Glu-113 of the red monomer shown in stick format.The distance between the hydroxyl group of Ser-178 and thebackbone carbonyl of Glu-113 is given. (C) Superimposition ofthe structures of the PCNA G178S mutant protein and wild-typePCNA (PDB entry 1PLQ). The distance between the backbonecarbonyl group of Glu-113 in the wild-type and mutant PCNAstructures is indicated.

13356 Biochemistry, Vol. 47, No. 50, 2008 Freudenthal et al.

most clearly seen by superimposition of the structures of thewild-type and mutant PCNA proteins (Figures 1C and 2A).

Loop J in the Wild-Type and Mutant Protein Structures.Altering the trajectory of the H1 and I1 antiparallel � sheetresults in structural changes in the extended loop betweenstrands H1 and I1 (residues 105-110), which we call loop J(Figure 2A). In the mutant protein structure, this loop adoptsa very different conformation in which the atoms of theprotein backbone have moved significantly from their posi-tions in the wild-type structure. For example, the R carbonof Lys-107 has moved 6.5 Å from its position in the wild-type protein structure. Figure 2B,C shows the proteinbackbone and electron density of loop J in its aberrantconformation in the mutant protein structure overlaid withthe protein backbone of loop J in its usual conformation inthe wild-type protein structure. On the basis of the clearelectron density of loop J in both the wild-type and mutantprotein structures, it is important to note that loop J is notmerely becoming more flexible and disordered in the mutantprotein structure. Instead, this loop has shifted from onestable configuration in the wild-type structure to anotherstable configuration in the mutant protein structure.

It should also be pointed out that the shift in loop J in thisstructure is not the result of crystal packing. The space groupand unit cell dimensions of our crystals are the same as thosereported for the wild-type protein (19), so observed structuraldifferences cannot be attributed to crystal packing. Moreover,in the crystal lattice, loop J does not contact any proteinsfrom neighboring asymmetric units; rather, it sticks out intosolvent-filled spaces of the crystal lattice (see Figure S1 ofthe Supporting Information).

Comparison of the Two PCNA Domains. To gain insightinto global structural changes occurring in PCNA as a resultof the G178S substitution beyond the affected loop J, wesuperimposed the protein backbone of the structures of thewild-type and mutant subunits (Figure 3). Domain B whichcontains the G178S substitution is structurally identical tothe wild-type form of PCNA. By contrast, domain A isaffected by the G178S mutation on the adjacent monomer.To quantify the degree of structural difference between thewild-type and mutant forms of PCNA, we calculated rmsdvalues between the backbone of the wild-type and mutant

forms of PCNA for each domain independently. The rmsdvalue for the domain which contains the G178S mutation(domain B) is 0.50 Å. The rmsd value for the domain withoutthe mutation (domain A) is 3 times as large at 1.3 Å. Thus,these results show that the G178S mutation is affecting thestructure of PCNA by acting in trans to alter the structureof the neighboring monomer’s domain A.

In summary, the only notable structural difference betweenthe wild-type and mutant forms of PCNA is in domain Awith the largest change being in the position of loop J. Thus,it is highly likely that the aberrant conformation of loop J isresponsible for the effect of this mutation on translesion DNAsynthesis.

Impact of the Mutant PCNA Protein on the ActiVity ofpol η. Genetic studies have shown that this mutant proteinblocks translesion DNA synthesis (25). To improve ourunderstanding of how the structural changes described hereinterfere with translesion DNA synthesis, we have directlymeasured the enzymatic activity of the nonclassical DNApolymerase pol η in the presence and absence of wild-type

FIGURE 2: Conformation of loop J in the wild-type and mutant structures. (A) Superimposition of the wild-type and PCNA G178S mutantprotein structures with the Ser-178 substitution and Glu-113 represented in stick format and the hydrogen bond between them shown asblack dots. The amino acid residues of loop J are indicated. (B) Close-up view of loop J showing the electron density (level ) 2.0) for thePCNA G178S mutant protein and the backbone of the wild-type and mutant proteins in ribbon representation. The distances between thewild-type and mutant protein backbone are specified. (C) Side view of loop J with the position of the amino acid residues indicated.

FIGURE 3: Superimposition of the PCNA monomer backbone ofwild-type and mutant PCNA proteins. The monomeric subunit islying on its side with the interdomain connector loop in the backto allow the separate domains to be easily viewed. The adjacentmutant monomeric subunit is colored blue with the G178Ssubstitution indicated. The G178S substitution, the site of monou-biquitination (Lys-164), and loop J are indicated. Domains A andB of the monomeric subunit are separated by a dashed line, andthe rmsd values were independently determined for each domain.


and mutant PCNA protein. Wild-type PCNA enhances theability of pol η to incorporate nucleotides opposite an abasicsite (22). Figure 4 shows the incorporation of pol η oppositean abasic site in a running start assay in the absence of PCNAand in the presence of either the wild-type or mutant formof PCNA. The presence of wild-type PCNA stimulates theability of pol η to incorporate a nucleotide opposite the abasicsite compared to that of pol η alone. By contrast, the mutantprotein appears to have no ability to stimulate incorporationopposite the abasic site.

To quantify the effects of wild-type and mutant PCNAproteins on the enzymatic activity of pol η, we measuredthe kinetics of incorporation of dGTP (the preferred incomingdNTP) opposite the abasic site under steady state conditions.The rate of incorporation was plotted as a function of dGTPconcentration (Figure 5); the Vmax and Km steady state kineticparameters were obtained from the best fit of the data to theMichaelis-Menten equation, and these parameters areprovided in Table 2. The catalytic efficiency (Vmax/Km) ofincorporation opposite the abasic site by pol η was repro-ducibly 2-4-fold greater in the presence of wild-type PCNAthan in the absence of PCNA, and this was due mainly to adecrease in the Km for nucleotide incorporation. By contrast,the catalytic efficiency was not greater in the presence ofthe PCNA G178S mutant protein than in its absence. Infact, the catalytic efficiency was reproducibly 2-4-fold lowerin the presence of the mutant PCNA than in its absence. Itshould be noted that like wild-type PCNA, the mutant PCNAprotein also decreased the Km for nucleotide incorporationin this context. The Vmax for incorporation in the presenceof the mutant form of PCNA, however, was ∼10-fold lowerthan that for incorporation in its absence. Thus, the mutantform of PCNA actually inhibits the ability of pol η toincorporate nucleotides opposite abasic sites.

The inhibitory effect observed with the mutant PCNAprotein requires the mutant protein to be loaded onto theprimer-template DNA. We showed this by omitting eitherreplication factor C (RFC, the ATP-dependent PCNA-loadingprotein) or ATP from the reaction so that the mutant PCNA

protein would not be loaded onto the DNA. In theseexperiments, the efficiencies of nucleotide incorporation bypol η were exactly the same as those measured when noPCNA was present. For example, when we omitted ATP,the catalytic efficiency of incorporation opposite the abasicsite in the presence of the unloaded mutant PCNA proteinwas 0.028, which is identical to the catalytic efficiency of0.029 determined in the absence of PCNA (Table 2). Thus,we observed no inhibition of pol η-catalyzed nucleotideincorporation opposite abasic sites when the mutant form ofPCNA was not loaded on the DNA substrate.

To determine whether the inhibition by this mutant PCNAprotein is specific to abasic sites, we used steady state kineticsto examine if the mutant form of PCNA could inhibit theincorporation of nucleotides opposite nondamaged DNA.Figure 6 shows the rate of dCTP incorporation opposite anondamaged G by pol η as a function of nucleotideconcentration. Here again, the catalytic efficiency of incor-poration opposite the nondamaged template was reproducibly2-4-fold greater in the presence of wild-type PCNA thanthat in its absence (Table 2). In this case, the increasedcatalytic efficiency in the presence of wild-type PCNA wasdue to both an increase in the Vmax and a decrease in the Km

for nucleotide incorporation in the presence of wild-typePCNA. The mutant PCNA protein inhibited the ability ofpol η to incorporate opposite the nondamaged DNA to a2-4-fold lower level of activity than pol η alone, and thiswas mainly due to an increase in the Km for nucleotideincorporation. Thus, this mutant protein inhibits nucleotideincorporation by pol η opposite both damaged and nondam-aged templates.

DISCUSSION

Interactions between PCNA and nonclassical polymeraseshave been shown to be essential for translesion synthesis.pol η possesses a PCNA-interacting peptide (PIP) motif atits extreme C-terminus (residues 621-628). While a pol ηmutant protein truncated at position 624 exhibits activity invitro, yeast expressing this truncated version of pol η havethe same defect in translesion synthesis as yeast completelylacking pol η (22). Thus, the interaction between PCNA andpol η is essential to pol η’s function in vivo. This may bebecause the interaction between PCNA and pol η leads toan increase in the catalytic efficiency (Vmax/Km) of nucleotideincorporation by pol η opposite damaged and nondamagedtemplates in vitro (22). Similarly, PCNA increases the DNAsynthesis activity of pol �, which lacks a PIP motif, on bothnondamaged DNA and UV-treated DNA (24). Incidentally,this also shows that there are functionally important interac-tions between nonclassical polymerases and PCNA that arenot mediated by PIP motifs.

Translesion synthesis by both pol η and pol � is severelydefective in yeast expressing the G178S mutant form ofPCNA. This was clearly demonstrated by experiments inwhich plasmids containing specific DNA lesions weretransformed into various yeast strains such that the trans-formation efficiency indicated the efficiency of DNA damagebypass (25). The reV6-1 strain (expressing the G178S mutantform of PCNA) bypasses both abasic sites and cis-synthymine dimers with an ∼1% efficiency compared to thatof the wild type (25). The reV3∆ strain (lacking pol �)

FIGURE 4: Running start experiment with pol η on an abasic site.(A) Schematic diagram of the 31/68-mer substrate used in therunning start assays with the ends of the template strand containingbiotin-streptavidin blocks. The X indicates the location of theabasic site. (B) Autoradiograph of the synthesis products after 5 or15 min following the addition of pol η. The arrow indicatesincorporation opposite the abasic site. Lanes labeled WT containthe wild-type PCNA protein, and lanes labeled MT contain thePCNA G178S mutant protein.


bypasses thymine dimers with an ∼94% efficiency comparedto that of the wild type; abasic sites are bypassed with an∼5% efficiency (25). The rad30∆ strain (lacking pol η)bypasses abasic sites with an ∼80% efficiency compared tothat of the wild type; cis-syn thymine dimers are bypassedwith an ∼15% efficiency (25). Only in the rad30∆ reV3∆strain are the efficiencies of bypassing either of these lesionsas low as they are in the reV6-1 strain (25).

To improve our understanding of why the G178S substitu-tion in PCNA leads to a loss of function of both pol η andpol �, but no other discernible effects on DNA replicationand cell growth, we determined the X-ray crystal structureof the mutant form of PCNA to a resolution of 2.9 Å. Wefound that while the global structures of the wild-type andmutant protein are the same, there is a significant difference

in the structure of a small region of PCNA near the subunitinterface. The substituted Ser-178 side chain forms a newhydrogen bond with the backbone carbonyl of Glu-113 onthe neighboring subunit. This new hydrogen bonds alters thetrajectory of the � sheet comprised of strands H1 and I1

(containing Glu-113). This has the effect of moving loop Jas much as 6.5 Å in the structure of the mutant protein fromits location in the structure of the wild-type protein (Figure2).

Since the alteration in loop J is the only significantdifference between the wild-type and mutant protein struc-tures, we conclude that the proper positioning of loop J iscritical for supporting translesion synthesis by both pol ηand pol �. Further support for this notion comes from ourstructural studies of the E113G mutant form of PCNA, which

FIGURE 5: Steady state kinetics of pol η on an abasic site. The rate of nucleotide incorporation was graphed as a function of dGTP concentrationfor (A) pol η alone, (B) pol η with wild-type PCNA protein, and (C) pol η with the PCNA G178S mutant protein. Autoradiographs of thegels showing the incorporation of a single dGTP across from an abasic site at the indicated concentrations of nucleotide are shown aboveeach graph. The solid lines represent the best fits of the data to the MichaelissMenten equation, and the Vmax and Km steady state parametersare given in Table 2.

Table 2: Steady State Kinetic Parameters of pol η-Catalyzed Nucleotide Incorporation

protein DNA Vmax (nM/min) Km (µM) Vmax/Km relative efficiency

pol η alone abasic site 1.0 ( 0.1 34 ( 8 0.029 1.0pol η with wild-type PCNA abasic site 0.70 ( 0.06 9.9 ( 3.0 0.071 2.4pol η with mutant PCNA abasic site 0.12 ( 0.01 12 ( 1 0.010 0.34pol η alone nondamaged 3.2 ( 0.2 0.67 ( 0.12 4.8 1.0pol η with wild-type PCNA nondamaged 4.8 ( 0.3 0.32 ( 0.07 15 3.1pol η with mutant PCNA nondamaged 3.4 ( 0.3 2.1 ( 0.4 1.6 0.33

FIGURE 6: Steady state kinetics of pol η on nondamaged DNA. The rate of nucleotide incorporation was graphed as a function of dCTPconcentration for (A) pol η alone, (B) pol η with wild-type PCNA protein, and (C) pol η with the PCNA G178S mutant protein.Autoradiographs of the gel showing the single incorporation of dCTP at the indicated concentrations of nucleotide are shown above eachgraph. The solid lines represent the best fits of the data to the MichaelissMenten equation, and the Vmax and Km steady state parameters aregiven in Table 2.


leads to a similar defect in translesion synthesis as does theG178S mutant PCNA protein (26). Glu-113 is at the subunitinterface directly across from Gly-178, and the side chainhydroxyl group of the G178S substitution forms a hydrogenbond with the backbone carbonyl of this residue. We haveobtained crystals of the E113G mutant form of PCNA andcollected X-ray diffraction data to a resolution of 3.6 Å.Comparisons of the electron densities at this resolution ofthe wild-type, G178S mutant, and E113G mutant proteinsshow that loop J is also in an aberrant configuration in thestructure of the E113G mutant protein as it is in the structureof the G178S protein (see Figure S2 of the SupportingInformation). Taken together, these data favor the hypothesisthat loop J of PCNA makes an important direct contact withnonclassical polymerases and that when loop J adopts anaberrant conformation in the case of these two mutantproteins, this contact is disrupted.

Additional evidence of the importance of loop J comesfrom previous structure-function studies of PCNA. Thesestudies have shown that several substitutions in and aroundloop J (residues 105-110) have been found to lead toincreased sensitivity to UV radiation and the DNA-damagingagent methyl methanesulfonate (MMS) (27). Cells expressingthe E104A/D105A double mutant have a severe defect intheir ability to grow following UV or MMS treatment (27).Cells expressing the K108A/D109A double mutant have aminor defect in their ability to survive MMS treatment (27).Cells expressing the D109A/R110A double mutant have aminor defect in their ability to survive UV and MMStreatment (27). These studies provide additional compellingevidence that loop J of PCNA is critical for translesion DNAsynthesis.

As described above, previous studies have shown that theinteraction between PCNA and pol η that is mediated bythe PIP motif of the polymerase is essential for translesionsynthesis (22). It is important to note that the binding sitefor the PIP motif is located on the opposite face of the PCNAring from loop J. The PIP motif binds to a hydrophobicpocket in domain B near the interdomain connector loop (35).Incidentally, we observe no significant structural differencesbetween the wild-type and G178S mutant PCNA proteinsin the regions of domain B or the interdomain connectorloop that binds the PIP motif. Loop J is located at the subunitinterface and extends out from the opposite face of the PCNAring. These observations imply that pol η can simultaneouslyinteract with both faces of the PCNA ring. The feasibilityof this model is supported by a structure of a complexcontaining the “little fingers” domain of E. coli pol IV, whichis a Y-family polymerase related to pol η, and the �-clampprocessivity factor, which is structurally similar to PCNA.The C-terminal peptide of pol IV (analogous to the pol ηPIP motif) binds in a pocket on one face of the �-clampring, and the little fingers domain of pol IV binds at thesubunit interface (36).

To improve our understanding of how the altered confor-mation of loop J of PCNA affects the function of pol η, wecompared the impact of the wild-type and mutant PCNAproteins on pol η’s activity. We found that this mutant proteinfails to stimulate the activity of pol η on both abasic sitesand nondamaged templates. Not only does it fail to stimulatepol η, this mutant form of PCNA actually inhibits the activityof pol η to levels 2-4-fold lower than that of pol η in the

absence of PCNA. As a result, the activity of pol η in thepresence of the PCNA G178S mutant protein is approxi-mately 10-fold lower than its activity in the presence of thewild-type PCNA protein. To the best of our knowledge, thisis the first amino acid substitution in PCNA that inhibits theactivity of a nonclassical DNA polymerase. It should bepointed out that the E113G mutant proteins also failed tostimulate the ability of pol η to incorporate nucleotidesopposite an abasic site, although no inhibition was observedwith this mutant protein (see Figure S3 of the SupportingInformation). It is unclear why the G178S mutant proteininhibits pol η and the E113G protein does not. One possibilityis that subtle differences in the positions of loop J betweenthese two mutant proteins account for this difference infunction. While the position of loop J is aberrant in thestructures of both mutant proteins, the position of the loopis closer to that of the wild-type protein in the E113G proteinstructure (see Figure S3 of the Supporting Information).

It should be noted that if RFC or ATP is omitted from thereaction, no inhibition of pol η is observed with the PCNAG178S mutant protein. This shows that the inhibition requiresthat the mutant PCNA protein be loaded onto the DNAsubstrate. The precise mechanism by which the G178Smutant form of PCNA inhibits the catalytic activity of pol ηis unclear, and this awaits a more detailed understanding ofprecisely how wild-type PCNA impacts the kinetic mecha-nism of nucleotide incorporation by pol η. However, onestraightforward possibility is that without the requisiteinteraction between pol η and loop J of PCNA, the presenceof PCNA on the DNA sterically interferes with the properbinding and positioning of pol η. This would prevent theformation of a productive polymerase-PCNA complex onthe DNA, which may be responsible for the defect intranslesion synthesis observed in cells expressing this mutantform of PCNA.

Finally, it had been suggested that the G178S substitutionmight block translesion synthesis by interfering with PCNAmonoubiquitination (25), which is required for translesionsynthesis (37). This is unlikely to be the case for severalreasons. First, the site of monoubiquitination, Lys-164, islocated in domain B of PCNA, which we have shown hereto have the same structure in both the mutant and wild-typeproteins. Second, it has previously been shown that theE113G mutant form of PCNA, which appears to blocktranslesion synthesis by the same mechanism used by theG178S mutant protein, is capable of being monoubiquitinatedin vitro by the Rad6-Rad18 complex (26). It should bepointed out, however, that any ability or inability of theG178S mutant protein to be monoubiquitinated is likely notrelevant to understanding the mechanism by which thismutant protein blocks translesion synthesis. This is becausethe data presented here show that this mutant PCNA protein,even in the absence of monoubiquitination, inhibits thecatalytic activity of pol η. This implies that the defect intranslesion synthesis caused by this mutant protein isindependent of its monoubiquitination state.

ACKNOWLEDGMENT

We thank Lokesh Gakher for providing technical as-sistance. We thank Lynne Dieckman, Christine Kondratick,and John Pryor for valuable discussions.


SUPPORTING INFORMATION AVAILABLE

Additional structural and kinetic data (Figures S1-S3).This material is available free of charge via the Internet athttp://pubs.acs.org.

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structure of a mutant form of proliferating cell nuclear antigen that blocks translesion dna...

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